sgrna-design-guide
sgRNA Design Guide
Overview
Selecting effective sgRNAs is the single most consequential decision in a CRISPR experiment. A poorly chosen guide RNA yields low editing efficiency, off-target mutations, or misleading phenotypes regardless of how well everything else is optimized. This guide provides a three-tiered decision strategy — validated sequences first, pre-computed designs second, de novo design only as a last resort — and explains the sgRNA quality metrics, PAM requirements, and application-specific targeting rules needed to make confident design choices.
The guide is adapted from the Biomni sgRNA Design Guide (Biomni Team, CC BY 4.0, November 2025) with expanded PAM rules, a unified decision framework, and additional best practices.
Key Concepts
PAM Requirements by Cas Enzyme
The protospacer adjacent motif (PAM) is a short DNA sequence the Cas nuclease requires for binding and cleavage. The PAM must be present in the genomic target immediately adjacent to the guide RNA binding site. Choosing the wrong enzyme or misidentifying the PAM renders a guide non-functional.
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